This invention relates to a novel human vascular permeability factor and to a cDNA clone representing the full size human vascular permeability factor protein.
Vascular permeability factors (VPFs) are proteins originally obtained from a variety of tumors which cause a rapid and reversible increase in blood vessel permeability when nanogram amounts are injected under the skin of a warm blooded mammal. VPF activity has been found in tumor ascites fluid from guinea pigs, hamsters and mice and is secreted by these tumors and a variety of tumor cell lines in vitro according to Senger et al., Science 219, 983-985 (1983).
In U.S. Pat. No. 4,456,550, a purified VPF is described which has the following characteristics:
(a) in an aqueous solution (0.01 M Na.sub.3 PO.sub.4, pH 7) whose concentration of NaCl is varied linearly, VPF is eluted from a heparin-Sepharose chromatography column in a peak centered at 0.4 NaCl; PA0 (b) in an aqueous solution of Na.sub.3 PO.sub.4, pH 7.0, whose concentration is varied linearly, VPF is eluted from a hydroxylapatite column in a peak centered at 0.25 M Na.sub.3 PO.sub.4 ; and PA0 (c) when subjected to SDS gel electrophoresis in a polyacrylamide slab gel (0.375 M tris-HCl, pH 8.8, 0.1% SDS) at 35 milliamps and 4.degree. C., VPF is localized to a region corresponding to a molecular weight between 34,000 and 45,000 daltons. PA0 (a) affinity chromatography with a column of heparin-Sepharose; PA0 (b) chromatography with a column of hydroxylapaptite; and PA0 (c) sodium dodecylsulfate/polyacrylamide gel electrophoresis. PA0 (a) it has a M.sub.r about 34,000-40,000 as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS/PAGE); PA0 (b) it is a disulfide-linked protein dimer; PA0 (c) it has a N-terminal amino acid sequence as follows: ##STR1## (d) it exhibits substantial mitogenic activity to endothelial cells in culture. PA0 (a) affinity chromatography of said conditioned culture medium with a column of heparin-Sepharose CL-6B; PA0 (b) cation exchange chromatography of the VPF active fractions from said affinity chromatography with a TSK SP-5-PW column; PA0 (c) high performance liquid chromatography (HPLC) of the VPF active fractions from said cation exchange chromatography with a Vydac C.sub.4 reversed phase HPLC column; and PA0 (d) HPLC of the VPF active fractions from said C.sub.4 HPLC with a Vydac C.sub.18 reversed phase HPLC column. PA0 (a) cation exchange chromatography of said conditioned cell culture medium, for example with a column of CM-cellulose, CM-Sephadex.RTM., Amberlite.degree. IR-120H or S-Sepharose Fast Flow cation exchanger; PA0 (b) metal affinity chromatography of the VPF active fractions from said cation exchange chromatography, for example with a Cu.sup.2+, Zn.sup.2+ or Ni.sup.2+ /iminodiacetic acid(IDA)/Sepharose column; and PA0 (c) reverse phase HPLC of the active VPF fractions from said method affinity chromatography, for example with a C.sub.4 or C.sub.18 reverse phase HPLC column.
VPF of the foregoing characteristics was thus purified about 1800 fold from serum-free conditioned medium of guinea pig tumor cell culture or 10,000 fold from ascites fluid by a series of steps consisting of:
According to said patent, as little as 200 ng (5.times.10.sup.-12 moles) of this purified VPF increased the vascular permeability equivalent to 1.25 .mu.g (4.times.10.sup.-9 moles) of histamine. Histamine is a standard permeability mediator described by Miles and Miles, J. Physiol. 118, 228-257 (1952). The VPF is said to have therapeutic value insofar as it enables blood nutrients to reach tissue with increased need for nutrients, as in wound healing.
According to Folkman and Klagsbrun, Science 235, 442-447 (1987), VPF causes leakage of proteins, including fibrinogen, from blood vessels, thereby initiating the formation of a fibrin gel which, in turn, may play a role in angiogenesis. See also Dvorak et al., J. Immunol. 122(1), 166-174 (1979); Dvorak, N. Engl. J. Med. 315, 1650-1659 (1986); Kadish et al., Tissue & Cell 11, 99 (1979); and Dvorak et al., J. Natl. Cancer Inst. 62, 1459-1472 (1979).
In copending application Ser. No. 07/87,739, filed Aug. 31, 1987, and assigned to a common assignee, a method of stimulating endothelial cell growth is provided which comprises subjecting said cells to a growth stimulating amount of a highly purified VPF. The highly purified VPF derived from guinea pig tumor cells has the following characteristics:
The foregoing highly purified guinea pig VPF, also referred to as gVPF, was isolated from serum-free conditioned culture medium of guinea pig tumor cells in a series of steps comprising:
In copending application Ser. No. 780, filed Sep. 2, 1988, and assigned to a common assignee, a method of producing antibodies against gVPF is provided in which certain peptide fragments of gVPF are used as immunogens.
Lobb et al., Int. J. Cancer 36, 473-478 (1985), describe a partially purified VPF from a human adenocarcinoma cell line HT-29 having a molecular weight of 45,000. This VPF, however, does not bind to immobilized heparin as does the VPF derived from guinea pig tumor cell material by Senger and Dvorak.
Senger et al., Cancer Res. 46, 5629-5632 (1986), describe the production of VPF from a variety of human tumor cell lines, namely human osteogenic sarcoma, bladder sarcoma, cervical carcinoma and fibrosarcoma cell lines. However, none of these human cell lines were found to be as active as the guinea pig cell line 10 for the producton of VPF.